Mutator activity and specificity of Escherichia coli dnaQ49 allele - effect of umuDC products

The high fidelity of DNA replication in Escherichia coli is ensured by the α (DnaE) and ɛ (DnaQ) subunits of DNA polymerase providing insertion fidelity, 3′ → 5′ exonuclease proofreading activity, and by the dam-directed mismatch repair system. dnaQ49 is a recessive allele that confers a temperature-sensitive proofreading phenotype resulting in a high rate of spontaneous mutations and chronic induction of the SOS response. The aim of this study was to analyse the mutational specificity of dnaQ49 in umuDC and ΔumuDC backgrounds at 28 and 37 °C in a system developed by J.H. Miller. We confirmed that the mutator activity of dnaQ49 was negligible at 28 °C and fully expressed at 37 °C. Of the six possible base pair substitutions, only GC → AT transitions and GC → TA and AT → TA transversions were appreciably increased. However, the most numerous mutations were frameshifts, −1G deletions and +1A insertions. All mutations which increased in response to dnaQ49 damage were to a various extent umuDC-dependent, especially −1G deletions. This type of mutations decreased in CC108dnaQ49ΔumuDC to 10% of the value found in CC108dnaQ49umuDC+ and increased in the presence of plasmids producing UmuD′C or UmuDC proteins. In the recovery of dnaQ49 mutator activity the plasmid harbouring umuD′C genes was more effective than the one harbouring umuDC. Analysis of mutational specificity of pol III with defective ɛ subunit indicates that continuation of DNA replication is allowed past G:T, C:T, T:T (or C:A, G:A, A:A) mismatches but does not allow for acceptance of T:C, C:C, A:C (or A:G, G:G, T:G) (the underlined base is in the template strand).